Mitochondria play key roles in many vital processes of a living cell. The list of such processes includes energy conversion (since the principle function of mitochondria is to provide the cell with energy), metabolism of certain substances (e.g. fatty acids), etc. Mitochondria are also directly involved in production and utilization of free radicals (FR) and reactive oxygen species (ROS)—extremely active compounds that can affect many processes within a living cell. Finally, mitochondria have been recently proved to play a key role in the process of programmed cell death.
Many diseases are known to be related to the dysfunction of mitochondria. This includes all disorders connected to increased production of FR and ROS, single or mass dying of cells within a tissue of an organ, dysfunction of programmed cell death mechanism (apoptosis), dysfunction of fatty acid metabolism etc.
It is assumed that by affecting mitochondria different aspects of functioning of the whole organism can be improved.
Within the framework of the invention a new method is developed to affect mitochondria within a living cell via targeted delivery and accumulation of biologically active compounds inside these organelles.
This approach possesses some obvious advantages. Targeted delivery of a compound allows to increase the efficiency of its application, to reduce the dosage (since the effective dose of the compound is achieved only inside the target compartment of the cell), to reduce probability and strength of side-effects.
The functional organization of mitochondria itself provides a unique opportunity for the targeting—functioning mitochondrion actively pumps-out protons from its matrix into cytoplasm. This process creates an extremely high electro-chemical potential of hydrogen ions (proton potential) on the inner membrane of mitochondrion.
Bioenergetic studies have resulted in finding of a number of compounds that can penetrate mitochondrial membrane and actively accumulate inside mitochondria in a proton potential-dependent fashion. These compounds were called “Skulachev-ions” (Green D. E., “The electromechanochemical model for energy coupling in mitochondria”, 1974, Biochem. Biophys. Acta., 346:27-780). Such ions usually do not reveal any significant biological activity. The main idea of the invention is to use “Skulachev-ions” to create new compounds that includes besides a “Skulachev-ion” some residue (in terms of the invention—effector moiety, or effector) which should be delivered into mitochondria.
At the moment a very limited number of mitochondrially-targeted biologically active compounds is known. Some related compounds are described in inventions U.S. Pat. No. 6,331,532 and EP 1047701 (mitochinol (MitoQ), Mitovitamin E (MitoVitE)) and EP 1534720 (superoxide dismutase and glutathione peroxidase mimetics, linked to an alkyl triphenilphosphonium). Some of these compounds are described in papers discussed below.
Compounds carrying superoxide dismutase and glutathione peroxidase mimetics are claimed in EP1534720 as mitochondrially targeted antioxidants suitable for treatment of diseases related to oxidative stress. In experimental examples that illustrate the invention EP1534720 it is shown that these compounds can penetrate mitochondrial membrane and accumulate inside mitochondria. Antioxidant abilities of the compounds were shown by studying of non-biological reactions (as chemical properties of substances or when interacting with isolated mitochondria in vitro). No data are presented on the effect of these mimetics upon the living cell and organism. Such mimetic compounds are most likely to interact with SH-groups of proteins. This can dramatically reduce efficiency and seriously limit possible application of mitochondrially-targeted antioxidants carrying mimetics of superoxide dismutase as well as glutathione peroxidase (so called ebselen), as has been shown by Filipovska A, Kelso G F, Brown S E, Beer S M, Smith R A, Murphy M P. J. Biol. Chem. 2005, 280(25):24113-26. This study has demonstrated that ebselen covalently linked to a mitochondria-targeting moiety (Mitoebselen) is of the same antioxidant efficiency as normal nontargeted ebselen. Therefore, even if mitoebselen is more active antioxidant comparing to ebselen, this positive property of mitoebselen is diminished by its undesired side activity.
Another substance claimed as mitochondrially targeted antioxidant is MitoVitE i.e. a compound containing triphenylphosphonium as targeting moiety and vitamin E as an antioxidant. Description of the invention EP 1047701 discloses data showing some antioxidant activity of this compound shown in rat brain homogenate as well as an ability of MitoVitE to penetrate isolated mitochondria and living cells in culture. It has been shown that 10 μM MitoVitE has no toxic effect on cells in culture. However, higher concentrations resulted in cell death. It should be noted that no antioxidant activity of MitoVitE on cells in culture, tissue or entire organism has been demonstrated.
The effect of MitoVitE on cell culture is described in publication Jauslin M L, Meier T, Smith R A, Murphy M P, FASEB J. 2003 17(13):1972-4. It was found that MitoVitE is able to prevent apoptosis in cultured cells; however, this effect is retained even in the presence of an uncoupler FCCP (3-fluoromethyl-carbonylcionide phenylhydrazone) that switches off targeted accumulation of MitoVitE in mitochondria. These data show that even if MitoVitE is targeted to mitochondria, such targeting does not play a significant role in its biological effect.
Mitochondrially targeted antioxidant MitoQ and its variants (MitoQ5, MitoQ3) consist of ubiqinon (ubiqinol in its reduced form) linked to triphenylphosphonium by C-10 linker (C5, C3 accordingly). In the description of invention U.S. Pat. No. 6,331,532, MitoQ is claimed as active compound for pharmaceutical compositions suitable for treatment or prophylactics of disorders related to oxidative stress. Experimental examples of this invention demonstrate antioxidant properties of MitoQ in cell-free systems, its ability to penetrate isolated mitochondria in vitro, effect of this compound on respiration of isolated mitochondria in vitro. However no data on the effect of MitoQ on living cells, tissue, organs of entire organism are presented in U.S. Pat. No. 6,331,532.
Some additional data can be found in PCT WO2005019233 of the same group of inventors, where they show that MitoQ is able to prevent lipid peroxidation of isolated mitochondria in vitro. Some other data are published in Adlam V J, Harrison J C, Porteous C M, James A M, Smith R A, Murphy M P, Sammut I A, 2005, FASEB J. 19:1088-95. In this study the authors have presented the only so far known example of MitoQ action on living organism in the experiment of feeding rats with MitoQ, followed by the study of their heart function with the Langendorff method (isolated heart perfusion). The data obtained indirectly support the claim that MitoQ can be used for prophylaxis or therapy of myocardial ischemic damage. However, several inaccuracies and arguable points of this study do not allow to consider this claim proved. In fact, the model used by the authors—30-minute normothermic ischaemia followed by reperfusion is a standard method to study ischaemic damage of myocardium. However, the major disadvantage of this method is the electric instability of the isolated heart during reperfusion. It is well known that a certain number of hearts cannot restore their activity due to periodical or constant fibrillation, and periodical arrhythmia is to occur in almost every heart during the experiment. Surprisingly, there is indication of neither fibrillation nor arrhythmia in this article. Therefore it remains uncertain, whether the results obtained characterize average properties of the whole group of experiments or only those experiments in which the arrhythmia was less pronounced. Besides, taking into account the above reasons, it is clear that the number of animals in each experimental group (six) is not sufficient for such a complicated experimental model.
The statement that the results presented in this work are not completely correct is partially supported by a rather strange observation of a significant increase in the contractile function in both control and experimental groups during reperfusion that should be followed by inevitable death of some cardiomyocytes. This result could be obtained if the calculation of the contractile function was performed using only active hearts, excluding “switched off” unstable ones, while, the rate of perfusion was calculated using all hearts. Such method is obviously incorrect. Whereas average results in MitoQ treated hearts are higher than control ones, these groups have not been compared directly and thus the significance of this difference is not clear.
Thus, the claim that MitoQ is a cardioprotector compound seems to be not convincing. Unfortunately there are no control group data on mitochondrial ultrastructure, lactate dehydrogenase loss from the heart, as well as measurements of cytochrome c release from mitochondria as well as caspase 3, complex I and aconitase activity.
In conclusion, accurate analysis of this study reveals some vulnerable points especially at stages of selection and analysis of obtained results. Most likely the authors are not very experienced with the model used. Therefore the cardioprotective action of MitoQ remains unproved.
It should also be noted that despite some promising observations concerning the MitoQ action, there are several results and calculations that cast doubt on the possibility of practical application of this compound. For example, it was shown in experiments with cell culture that MitoQ provides its antioxidant and anti-apoptotic action at concentration about 1 μM in the medium. It means that MitoQ concentrations inside the mitochondria should be 1 mM provided that membrane potential is about 180 mV. On the other hand, it was shown by Smith R A, Porteous C M, Gane A M, Murphy M P, Proc Natl Acad Sci USA, 2003, 100(9):5407-12 when feeding MitoQ to animals, the accumulation of MitoQ in the most oxidative stress sensitive tissues (heart and brain) amounts up to 100 pmoles/gram, which corresponds to the MitoQ concentration inside heart mitochondria equal to 100 nM. This is more than 1000 times lower than the concentration shown to be efficient in the cell culture experiments. Formally, the concentration can be raised by the increase of amounts of MitoQ fed to animals, but this is, in fact, not possible due to the toxicity of the compound.
Summarizing the data above, all the mitochondrially targeted compounds disclosed so far are mitochondrially targeted antioxidants. No other mitochondrially-targeted biologically-active compounds are known to date. It should be remarked that the described substances claimed as mitochondrially targeted antioxidants cannot be applied yet to the claimed purposes and their perspectives are unclear due to the lack of information on their biological activity, especially taking into account that most of them have already been proved inefficient or toxic.